Extract
Note: Please read the complete
full text with Figures and Tables at
amplitude were calculated to describe the changes in myoelectric
activity of the SO. The frequency of SPSO is given as
mean±SEM, and the amplitude was calculated as the range
in the same period. Statistical comparisons were carried out
by using Student’s t-test with SPSS (version 10) software,
as appropriate. A minimum P value of <0.05 was selected to
determine significance.
Results
Basal myoelectric activity All experiments started with a
control recording of basal myoelectric activity for 10 min,
during which time original activities of both the duodenum
and the SO were observed to ensure that the myoelectric
activity of the SO was different from that of the duodenum.
To obtain original activities of the duodenum, these signals
were not digitally high-pass filtered. In the control
recording of basal myoelectric activity, single, regular SPSO with
frequencies ranging from 0.17 to 0.88 Hz (mean frequency,
0.45±0.15 Hz, n=78), amplitudes of 65_257 µV, and maximum
potentials of 320_350 µV were observed (Figure 1).
Excitatory effects of orexins on myoelectric activity of
the SO The frequency and amplitude of SPSO after
intravenous injection of OXA (4 nmol/kg) were obviously increased
in 0.5 min after injection (n=6, P<0.01) relative to the same
parameters after the injection of saline (Figure 2A, 2D). In
some experiments (n=3), SPSO clusters with 4_7 single SPSO
in each were observed. Also, intravenous application of 4
nmol/kg OXB had markedly excitatory actions on the
myoelectric activity of the SO (n=6,
P<0.01) (Figure 2C, 2D). As shown in Figure 2D, the response induced by OXA or OXB
(4 nmol/kg) rose to a peak rapidly during the first 3 min, and
then declined and gradually approached the control level
during the subsequent 9 min. Furthermore, the myoelectric
activities of the SO were tested with 4 different
concentrations of orexins, and their effects were found to be
dose-dependent. As illustrated by the concentration-response
curve presented in Figure 2E, 4 nmol/kg OXA produced
potent effects on the myoelectric activity of the SO
(n=6, P<0.01), whereas 2 nmol/kg OXA produced a weaker response
(n=6, P<0.01). Doses of 0.5 nmol/kg and 1 nmol/kg had no effect
(n=6, P>0.05, respectively). Similarly, OXB also
dose-dependently activated the myoelectric activity of the SO (data
not shown). Therefore, these data indicate that orexins can
exert a dose-dependent excitatory effect on the myoelectric
activity of the SO.
Local injection of 10 µL saline (0.9%) had no significant
influence on the frequency or amplitude of SPSO. In
contrast, the frequency was obviously increased after local
application of 0.4 nmol OXA (n=6, P<0.05) (Figure 2B) or 0.4
nmol ORB (n=6, P<0.05) (data not shown).
These results indicate that both intravenous and local
injections of orexins produce modulatory effects on the SO.
Antagonistic effects of atropine on the excitatory effects
of OXA Because the SO is richly innervated by cholinergic
neurons and the enteric cholinergic system plays an
important role in the regulation of SO motor
activity[15], we hypothesized that the effects of orexins on SO motility might
be associated with cholinergic modulation. To investigate
this, we further tested the actions of atropine, a muscarinic
receptor antagonist, on the orexin-increased myoelectric
activity of the SO. First, when atropine (0.1 mg/kg) was
intravenously administered alone, a complete antagonistic effect
on the myoelectric activity of the SO was observed
(n=6,
P<0.01) (Figure 3A). Intravenous administration of atropine
(0.1 mg/kg) also fully antagonized the excitatory effects of
OXA (n=6, P<0.01; Figure 3B, 3C) and OXB
(n=6, P<0.01; Figure 3C) on the myoelectric activity of the SO. These
observations thus strongly imply that the excitatory effects
of orexins on the myoelectric activity of the SO are partially,
at least, dependent on cholinergic pathways.
Discussion
The orexin system is generally considered to control food
intake at the level of the hypothalamus. Interestingly, it has
been shown that orexins and orexin receptors (OX-1R and
OX-2R) are also expressed in neurons and endocrine cells in
the gut[6,16,17]. Recent studies suggested that orexins, like
5-hydroxytryptamine (5-HT), vasoactive intestinal peptide
(VIP) and several other gut hormones, could modulate
gastrointestinal motility[5,7,18_20]. Given that the SO plays an
important role in the digestive system, we hypothesized that
orexins might play a role in modulating the motility of the SO.
In the present study, we investigated the effects of
peripheral orexins on the SO in fasted rabbits by recording
the myoelectric activity of the SO. In fasted rabbits, regular,
single SPSO with frequencies of 0.17_0.88 Hz and amplitudes
of 65_257 µV describes the basic rhythm of myoelectric
activity of the SO. A dose-dependent excitatory effect of orexins
on the frequency of SPSO was observed when either OXA
or OXB was given at 4 different concentrations. Moreover,
local administration of OXA or OXB also obviously increased
the myoelectric activity of the SO.
Although we did not visualize changes in the pressure
in the bile duct in the present study, the marked and
continually increased myoelectric activity might indicate
contractility of the sphincter[21]. This means that orexins appear to be
potential stimulators of SO motility, resulting in a reduction
in trans-sphincteric flow. Thus our findings have
broadened the possible roles of the peripheral orexin system in the
modulation of the digestive system. Nevertheless, the
actual physiological role of the orexin system in the regulation
of the SO needs to be further clarified.
Although orexins were administered peripherally in the
present study, it is unclear whether orexins excite the SO in a
peripheral or central manner. Morphological evidence
indicates that orexin receptors and fibers are distributed in dosal
vagal complex that plays a central role in controlling gas
trointestinal function and comprises the dorsal motor nucleus
of the vagus (DMN) and the nucleus of the solitary tract.
Studies have suggested that orexins in the DMN have
potent effects in promoting gastric
motility[7,22,23]. Therefore, it is possible that orexins applied intravenously in the present
study may stimulate the DMN after crossing the blood-brain
barrier[24,25], which would subsequently increase the
myoelectric activity of the SO. Furthermore, local administration
of orexins (clearly not centrally) also evoked an increase in
myoelectric activity. This implies that the peripheral pathway,
in addition to the central pathway, may also be responsible
for the effects of orexins on SO motility.
We next sought to elucidate the possible mechanisms
underlying the effect of OXA on the myoelectric activity of
SO. As we know, there is a vagal cholinergic excitatory
pathway that, when active, has an important role in stimulating
gastric function[26]. Thus we hypothesized that the increased
motility of the SO caused by orexins might involve the
enteric cholinergic neurons. To address this point, we injected
the rabbits with 0.1 mg/kg atropine (a cholinergic muscarinic
receptor antagonist) after intravenous injection of OXA or
OXB. We found that atropine completely antagonized the
orexin-increased myoelectric activity, indicating that the
activation of the vagal cholinergic pathway is required for the
stimulation effect of orexins on SO motor function. Our
results are consistent with those of previous reports that have
demonstrated that OXA can evoke the release of
acetylcholine (ACh) from the enteric cholinergic neurons due to
stimulation of the OX-1R, and then cause contractions of guinea
pig ileum[27]. Additionally, we found in the present study
that systemic administration of atropine alone blocked the
myoelectric activity of the SO. Therefore, we can postulate
that both basal electric activity and orexin-evoked electric
activity of the SO may be mediated by cholinergic modulation.
However, due to a lack of morphological data, our work
cannot exclude the possibility that orexins directly regulate SO
motility by binding to orexin receptors in SO tissue. For this
reason, additional studies are needed to determine if orexins
and orexin receptors are expressed in the SO area.
In summary, in general, peripheral OXA exerts excitatory
effects on SO. It seems likely that the orexin system may
play a significant role in modulating the function of SO, in
part through activation of the cholinergic pathway.
Acknowledgement
We thank Hu-cheng LI, Hong-xiao FAN and Hong-mei
Xu for their technical assistance.
References
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